Anti-viral isolates obtainable from leeches

ABSTRACT

Methods substances and composition derived from leeches, bacteria associated with leeches, or combinations thereof are provided to prevent retroviral infections of host cells. More particularly, the invention relates to the inhibition of HIV replication. Thus the invention provides methods and substances for treating and/or preventing HIV infection and/or AIDS. The compounds or mixtures are obtainable by solvent extraction techniques and separation steps and were tested in the following manner. Two syncytium-forming HIV isolates HIV AMS55 and HIV Ams37 were inoculated in PBMC and monocytes. During 14 days HIV induced cytopathic effects (CPE) (syncytium formation) and the virus production as determined by a p24 capture ELISA was monitored, after addition of leech head extracts. The results of these tests with the partly purified HIV inhibiting substance or substances is as follows: 3 fractions inhibited both syncytium formation and p24 antigen by 100% at a concentration of: 0.67 μM, 0.5 μM and 0.17 μM respectively in both HIV isolates. A fourth fraction underwent dilution series and inhibited both CPE and p—24 antigen 100% from 0.5 μM upwards in both HIV isolates.

The present invention relates to the field of anti-viral compounds, e.g.in the field of immune diseases, particularly acquired immune diseases,more in particular to infections with human immunodeficiency virus.

A new human immunodeficiency with opportunistic infections andmalignicies was described in 1981. The causative agent was found to be aretrovirus, Human Immunodeficiency Virus (HIV). The pathogenesis of thedisease begins with the alteration and eventual depletion of T4lymphocyte cells, leading to a progressive destruction of the immunesystem.

HIV is a retrovirus, consisting of a capsulated RNA-genome surrounded bya double-layer core-membrane. An early finding was the variation ofnucleotide sequences of certain parts of the genome, especially in theenvelop gene (Shaw, et al, 1986).

the gag and pol genes of HIV are translated as two polyproteins (Pr55gagand Pr160gag-pol) (Jacks, et al, 1988). These are subsequently cleavedby the action of a virus-encoded protease into four structural gagproteins of the virion core (p17, p24, p7 and p6) (Veronese, F D., etal., 1987), and the pol-encoded enzymes essential for retrovirusreplication (protease, reverse transcriptase, ribonuclease H., andendonuclease).

Viral core-glycoprotein precursor gp160 component gp120 binds to CD-4, acellular receptor of the susceptible T-cell subset with high affinity(Berman, et al, 1989). Therefore, viral recognition and fusion of CD-4with gp120 is a pivotal occurrence in the infection cycle. It should benoted however, that other factors may play a role as well: Neutralizingantibodies to the V3 loop of gp120 prevent infection even though they donot inhibit binding to CD4 (Rusche, et al, 1985), and

Productive infection of monocytes, in absence of proliferation and DNAsynthesis of T lymphocytes, can occur and serve as relative long-livedreservoirs of virus (Weinberg, et al, 1991).

Drug development against HIV infection sofar has aimed at preventingfusion between the CD4 membrane protein and viral gp120 and thuspreventing mediation of HIV entry into the cytoplasm of T4 lymphocytes.Vaccination with recombinant gp120 (rgp120) led to protection ofchimpansees from HIV infection (Berman P W., et al, 1990). Cytopathicfusion of CD-4 cells (syncytia) occurs on expression of gp120 at theplasma membrane. Soluble CD4 blocks secretion and surface expression ofgp120 prevents such cell fusion (Buonocore & Rose, 1990).

Other attempts to develop drugs against infection with HIV have so farfocused on the inhibition of HIV reverse transcriptase (RT) and HIVprotease. RT is the target of AZT, the first anti-HIV drug in clinicaluse. A TIBO derivative RS2150 was found to inhibit 50% of HIV RT at aconcentration of 3.1 μM and 4.9 μM respectively for different viralstrains (Pauwels, et al, 1990).

HIV protease inhibition is for example disclosed in EP 541168, U.S. Pat.No. 5,196,438, and in WO092/08701. Inhibition of proteases results inthe production of non-infectious virus particles and unprocessed gag andgag-pol precursor proteins (Kohn, et al, 1988: Loeb, et al, 1990).Recent disclosures aiming at inhibition of either HIV protease or HIV RTin patent literature are: WO 95/20384, WO 95/16688, WO94/06454, EP0666755, EP 0666842, WO 95/14011, and many more.

A range of antigenically distinct HIVs have been isolated, (even withinone individual) causing a major problem in drug development against HIV.Moreover, many drugs which seem promising in vitro suffer from the factthat the virus is capable of escaping their effect in vivo by quickmutagenesis.

The present invention provides a new group of substances capable ofinhibiting Human Immunodeficiency Virus, which can thus be used toprevent or combat HIV infection and/or AIDS.

During laboratory testing of a newly discovered range of proteaseinhibitors from leeches (see EP 94117053.2 and EP 95103637.5) verypotent elastase-, chymotrypsin-, trypsin-, plasmin- and thrombininhibitors were defined. Since the proteolytic cleavage of the gag andenv precursors in the replication cycle of HIV is an important step, itwas investigated if the newly described inhibitors would have any effecton such virus replication. Development of inhibitors is a rationale inanti-viral drug development. The aim of such study was to evaluate theantiviral capacity of the protease inhibitors, which were the subject ofEP 94117053.2 and EP 95103637.5, on primary cells.

It was concluded in the above-mentioned patent applications, thatanti-viral capacity was indeed present in the described inhibitors.However, it has now been surprisingly found, that a farm more potentanti-viral substance or group of substances resided in the originalleech and leech-head ethylalcoholic extractions. These substances orgroups of substances containing such surprising activity are the subjectof the present invention. We have purified and characterized thesubstances.

The present invention thus provides an isolate or compound havinginhibitory activity towards virusses, e.g. towards a humanimmunodeficiency virus, obtainable from leeches or organisms associatedwith leeches of the phylum uniramia by solvent extraction techniques andhaving a molecular weight of about 300-600, preferably about 400-500Dalton. Preferably the compound is obtainable from the subclass ofeuhirudinae, in particular it is obtainable from the suborderhirudiniformes, preferably the order of arhynchobelidae. Most preferredare compounds which are obtainable from the family hirudinidae, inparticular those which are obtainable from the genus Limnatis, inparticular the species L. nilotica. L. nilotica (Savigny, 1820, asfollowing from Autrum 1936) is described as a “nasal leech” or horseleech (Mouquin-Tandon 1846). It was found to be present in the wholelittoral area of the Mediterranean (Harant, 1928; Jarry, 1959). It livesin spring fountains and “oueds”, and feeds on cattle, dogs and man(Blaise, 1874/5; Neveu & Lemaire, 1938; Turner, 1969; Keegan, et al,1970).

Amazingly, the feeding habits of this leech differ from otherhaemotophaguos leeches. It remains attached to its host (nasal—andlaryngeal cavity) for prolonged periods (weeks to months). The animalfeeds on its host repeatedly. We have observed that drinking cattle wereinfected with these leeches, which did not drop off while the cattle aredrinking water. Only thick, fat, adult-size leeches do drop off at suchoccasions. Therefore, it is clear that this species of leech is mostlyfree of antigenic or immunogenic substances in its mucus or salivarygland productions. Reports of host animals dying from this species ofleech mention anemia as the one common cause, but no direct antigeniceffect has been described to our knowledge. In one aspect the inventionthus provides compounds having HIV inhibiting activity obtainable fromthe leech Limnatis nilotica or fragments of derivatives of suchcompounds having similar activity.

Such compounds can be derived from all body parts and secretions of theleech, including saliva- and gut-, intestinal- and skin secretions andmucus.

The most suitable source for the compounds according to the invention isprobably solvent extraction of the heads of the leeches, or compoundsassociated with these heads. As may be understood it is also possiblethat the actual source of the compounds according to the invention maynot be the leech itself, but it may be derived from a leech associatedmaterial or organism, such as a bacterium.

These compounds obtainable from for instance a bacterium associated withthe leeches are also a part of the present invention. The requirement isthat they are obtainable by solvent extraction techniques when appliedto the leeches, that they have the characteristics as disclosed herein,of which the anti-viral activity, and esp. the HIV-inhibition, is themost important characteristic. The compounds may even be the result ofinteraction between leech derived and other materials.

The present invention also provides a method for obtaining a compoundhaving human immunodeficiency virus inhibiting activity comprisingdehydrating leech tissue, preferably leech heads, or secretionspreferably saliva, optionally lyophilizing the resulting extract,suspending the lyophilized material in an aqueous solution, centrifugingthe material to obtain a pellet, loading resulting supernatant on a sizefractionation column, eluting the column with an aqueous solution,collecting fractions of the eluted material, testing the fractions forhuman immunodeficiency virus inhibiting activity and collecting theactive fractions.

Isolates obtained by the process of the invention undoubtedly posses ananti-viral activity. Without being wished to be bound to a theory, it isassumed—e.g. on the basis on the analytical test data herein-below—thatthe anti-viral activity can be attributed to an active ingredient havinga general formula Ar¹-spacer-Ar², wherein Ar¹ is an aromatic six-ring,which preferably is para-substituted, e.g. with a hydroxyl group or anamino group, which siz-ring can also be substituted at the otherpositions with e.g. C₁₋₄ alkyl groups, and halogens; wherein the spacercontains a C₃₋₈ alkylene group coupled through a hydrophilic group, suchas an amido group, to Ar¹; and wherein Ar² is a heteroaromatic2-ringsystem based on carbon and nitrogen atoms (the 2-ringsystem having9 or 10 atoms (purine- or naphthene-like ringsystems)), which ringsystemcan be substituted with e.g. C₃₋₈ alkyl groups, hydroxy groups, aminogroups, halogens etc; as well as addition salts, solvates and dimers ofthese isolates.

It can however not be ruled out that other ingredients of the isolatesobtained are the potent inhibitors or anti-viral compounds or attributeto the activity shown.

Preferably, such a method further comprises subjecting active fractionsto a further separation step, preferably using an HPLC column. Theisolates or compounds obtainable by these methods which have beenisolated sofar have a low molecular weight. When testing these compoundsfor their inhibitory activity, it was found that the compounds ormixtures of compounds according to the invention have anti-viralactivity in general, e.g. human immunodeficiency virus inhibitoryactivity in the micromolar range. It is e.g. found that the isolatecompletely inhibits human immunodeficiency virus induced syncytiumformation of p24 production at 0.5 μM, preferably 0.17 μM, mostpreferably 0.067 μM concentrations. The invention further provides theuse of these compounds or mixtures in preventing or combating infectionswith HIV an/or AIDS and thus also provides a pharmaceutical compositioncomprising at least one compound according to the invention and asuitable vehicle for administration. Based on the kind and amount ofactivity of the compounds and the amounts tolerated, which can bedetermined in animal studies using rising doses the person skilled inthe art will be able to develop these pharmaceutical formulations.

Administration may be accomplished in any suitable manner, although forsome compounds in systemic applications parenteral routes may bepreferred. Dosages for these substances can be taken from the literatureand designed on the basis of specific activities of the substances, themolecular weight of these substances, the weight of the subject to betreated, the kind of application etc. Dosage will usually lie between0.1 μg/kg and 10 mg/kg bodyweight. Suitable excipients are well known inthe art and range from water for injection to carrier proteins such asserum albumin for e.g. lyophilized preparations and inert substancessuch as mannitol, cellulose and dextran for tablets or granulates. Theeasiest way is of course simply providing the compounds in sterile water(or saline) for injection.)

Included in the invention are also mutations, isoforms, derivatives,such as salts and solvates, fragments or even peptidomimetics or othermimicks, and anti-idiotype or catalytic antibodies of the HIV(replication) inhibiting substances.

Synthetic manufacture of the invented substances, mutations, isoforms,derivatives, and fragments is also included in this invention, as areintermediates which are useful for obtaining the compounds according tothe invention.

Experimental

During research work with L. nilotica derived protease inhibitors, itwas surprisingly observed, that, next to HIV replication inhibitionactivity of the protease inhibitors mentioned hereinbefore, anotherfraction, obtained from L. nilotica heads ethanol extracts, was far morepotent. This group of substances is named SILIAVIR in the presentdescription.

In a biologic test system with primary cells, which is described furtherhereinbelow, it was concluded, that whole leech's head'extract showed adose dependent inhibition of HIV replication in two separate HIVisolates, being almost complete at a concentration of 0.5 μM (see Table1 and Table 2 of FIG. 1).

Further isolation and purification methods in steps, finally led to agroup of active compounds also called substances herein, having amolecular weight of around 05. kD, which show e.g. HIV replicationinhibition at 100% at such low concentrations as 0.06 μM.

The biological effects as measured are not yet related to one of theknow strategies in drug development against viruses, in particularretroviruses, and more specifically against HIV. However, any of thesewell-known strategies could be involved (e.g. RT inhibition, sCD4, gp120substitute, HIV protease inhibition). Other possible ways of activationare not excluded) for example, but not limited to: effects on the cells,as well as effects on the challenging virus, or the combination of thetwo), and are being researched.

The present invention reveals the isolation and purification steps, incombination with the biologic test system in primary cells, leading tothis group of new and unique substances, having the specific activitiesas described. Applicant specifically intends that this substancesSiliavir as invented, and described herein, includes such substancehowever produce, be it through sequential and block synthesis, orthrough gene cloning and expression, or by whatever other syntheticmechanism.

Description of Biologic testing and of Isolation and Purification.

A. Biologic test system

Peripheral blood mononuclear cells

Phytohaemagglutinin (PHA) stimulate PBMC from healthy donors wereinoculated with two different HIV isolates (HIVAms 37 and HIVAms 55).After a two-hour exposure to the HIV isolates, the inoculum was removedand serial concentrations Siliavir were added to the cultures. Mediumwas changed twice a week, fresh PMA-stimulated PBMC were added everyweek. Buffy-coats are routinely screened for viral contaminants and usedonly when negative for these contaminants. Virus production in thecultures was monitored with a p24-capture ELISA, detecting p24coreprotein of HIV. Cultures were also monitored for the occurrence ofcytopathic effects (syncytium formation).

Culture medium

During the first two days after isolation of the cells, primaryperipheral blood leukocytes were cultured in Iscove's ModifiedDulbecco's Medium (IMDM), supplemented with 10% foetal Calf serum (FCS),polybrene (5 μg/ml), phytohaemagglutinin (5 μg/ml), penicillin (100IU/ml) and streptomycin (100 IU/ml). The T-cell blasts were then furthercultured in IMDM supplemented with 10% FCS, polybrene (5 μg/ml),recombinant IL-2 (10 IU/ml), penicillin (100 IU/ml) and streptomycin(100 IU/ml).

Viruses

High titre inocula of two primary syncytium inducing HIV isolates wereprepared. Titers of stock were determined in a TCID50 assay.

During the first two days after isolation of the cells, primaryperipheral blood leukocytes were cultured in Iscove's ModifiedDulbecco's Medium (IMDM), supplemented with 10% foetal Calf serum (FCS),polybrene (5 μg/ml), phytohaemagglutinin (5 μg/ml), penicillin (100IU/ml) and streptomycin (100 IU/ml). The T-cell blasts were then furthercultured in IMDM supplemented with 10% FCS, polybrene (5 μg/ml),recombinant IL-2 (10 IU/ml), penicillin (100 IU/ml) and streptomycin(100 IU/ml).

Experimental design

A total of 10⁷ PHA-stimulated PBMC were inoculated with 10⁴ TCID50/ml ofthe primary HIV isolates in a volume of 1 ml for 2 hours at 37° C. After2 hours, cells were washed in a total volume of 30 ml. Aftercentrifugation, supernatant was discarded to remove non-absorbed virus.Cells were subsequently resuspended to a final concentration of 10⁶/ml.From each cell suspension 100 μl aliquots containing 10⁵ cells weretransferred to wells of a 96 well tissue culture plate. Dilutions offractions of Siliavir were made from stock solutions of 1 μM, in culturemedium in such a way that after addition of 50 μl to each well the finalconcentrations in the wells was 0.063, 0.125 μM, 0.25 μM, and 0.5 μM.Cells that received only medium served as untreated control cultures.Each concentration was analyzed in four fold. Cells were cultured in ahumidified atmosphere at 37° C., 5% CO₂. Addition of 10⁵ fresh PHAstimulated PBMC and medium was performed on day 7. In parallel, a newdose of the same concentrations of Siliavir was added.

Controls

On days 4 and 7, cultures were analyzed for any HIV-induced cytopathiceffect as reflected by the presence of syncytia. At days 7, and 14, 30μ1 of the culture supernatant was harvested to analyze the presence ofp24 antigen in a p-24-antigen-capture-ELISA. For this, twice a week, 30μl aliquots were harvested from the cultures and inactivated by theaddition of 30 μl 0.2% Triton-X-100. 15 μl of this mixture was added towells of a 96 well ELISA plate coated for 2 hours at 37° C. with ananti-p24 antibody shown to recognize all HIV isolates. Antigen wasallowed to bind during 2 hours at 37° C. Bound p24 was detected withimmunoglobulin (90′ at 37° C.). Then, substrate (TMB) was added andafter 20′, the reaction was stopped by the addition of H₂SO₄.

Results are presented in Tables 1 and 2 (FIG. 1). The tables should beread with the following in mind.

Calculation of results

For the calculation of the percentage inhibition the following formulawas used ${{Percentage}\quad {inhibition}} = {\frac{\begin{matrix}{{p24}\quad {production}\quad {in}} \\{{untreated}\quad {cultures}}\end{matrix} - \begin{matrix}{{p24}\quad {production}\quad {in}\quad {cultures}} \\{{exposed}\quad {to}\quad {substance}}\end{matrix}}{{p24}\quad {production}\quad {in}\quad {untreated}\quad {cultures}} \times 100\quad \%}$

Criteria

Cultures were considered positive if:

Syncytia formation was observed on at least one occasion (cultures wereexamined twice a week) in combination with an elevated p24 antigencontent in the supernatant on at least one occasion.

Syncytium score:

− no syncytia observed in any of the four replicate cultures,

± syncytia observed in one out of four replicate cultures

+ syncytia observed in two out of four replicate cultures

++ syncytia observed in three out of four replicate cultures

+++ syncytia observed in four out of four replicate cultures

Results

HIV-1 induced cytopathic effects (CPE), production of p24 antigen, andcalculated percentages inhibition are demonstrated in Tables 3 and 4 forsubstance 1 and in Tables 1 and 2 for substance 3.

Data for p24 production represent the mean of four replicate cultures

Cultures inoculated with 10² TCID_(to) remained negative for virusproduction.

Substance three, mentioned in these tables, represents whole leech headsextract. The extract processing, purification and isolation aredescribed below.

Each time a new purification step takes place (see below), the describedbiologic test system is used as an “affinity test-system” to furthernarrow the active substances from the extract fraction.

B. Methods for isolation and purification

A total of 15 g of cut heads from frozen Limnatis nilotica weredehydrated in 94% ethylalcohol at room temperature. Three changes of atotal of 134 ml, after which 400 ml destilled water was added, and theextract was subsequently lyophilized in vials. A total of ca. 90 mg oflyophilized material was assembled. (One can also use, as analternative, chopped heads of these leeches, or use activated mucussecretions from live leeches, by immersing them for instance for 10minutes in 4% ethylalcohol at room temperature).

The lyophilized material (ca. 90 mg) was first suspended in 4.55 mldestilled water. Thereafter, three portions of 4.55 ml of destilledwater were added. 50% of this suspension (9.1 ml) was further dilutedwith 30 ml destilled water, the other 50% stored at −20° C. thesuspension was divided over two centrifugation tubes and centrifuged.The pellet was stored at −20° C. 50% of the supernatant was also storedat −20° C. The other 50% of the supernatant was loaded on a SephadexG-75 column (length 180 cm, diameter 1.85 cm). Eluens is distilledwater, fraction size: ca. 4-7 ml; flow adjusted at ca. 0.5 to 1 ml/min.A total of 149 fractions were sampled and absorption was measured at 214nm. The flow at the column reduced from its initial setting of 1 ml/minto ca. 0.5 ml/min and the fraction size diminished from initial 7 ml to4 ml (fraction 1 to 25: ca. 7 ml; fraction 26 to 29: ca. 6 ml; fraction30 to 79; ca. 5 ml and fraction 80-149; ca. 4 ml). A slight opalescensewas visible in fractions 19 to 24, while a precipitation was visible infractions 95 to 108. Based on the absorption diagram (FIG. 2) andSDS-PAGE (FIG. 3) the following pools for further trials were made:

fraction 16 to 29 (ca. 83 ml); sample name: 3A

fraction 30 to 75 (ca. 230 ml); sample name: 3B

fraction 76 to 97 (ca. 92 ml); sample name: 3C

fraction 98 to 130 (ca. 132 ml); sample name: 3D.

The precipitation found in pool 98 to 130 is stored at −20° C.

Total material through gel filtration is appr. 50%×50% of 90 mg=appr.22.5 mg.

Samples A through D were presented for testing with the biologicaltesting system using primary cells as described, each sample estimatedat 5 mg (incl. losses).

Estimation of weight of substance of the samples, is as follows:

Sample 3A: 83 ml, containing 5 mg, concentration 60 μg/ml.

Sample 3B: 230 ml, containing 5 mg, concentration 22 μg/ml.

Sample 3C: 92 ml, containing 5 mg, concentration 54 μg/ml.

Sample 3D: 132 ml, containing 5 mg, concentration 38 μg/ml.

From Sample 3A a fraction of 0.625 ml was added to 2.9 ml destilledwater, 1 ml of 5 times concentrated Iscove's Modified Dulbecco's medium(IMDM) and 0.5 ml Foetal Calf serum (FCS), thus resulting in a solutionwith a concentration of 1 μM.

From Sample 3B a fraction of 1.7 ml was added to 1.8 ml destilled water,1 ml of 5 times concentrated IMDM and 0.5 ml FCS, thus resulting in asolution with a concentration of 1 μM.

From sample 3C a fraction of 0.694 ml was added to 2.8 ml destilledwater, 1 ml of 5 times concentrated IMDM, and 0.5 ml FCS, thus resultingin a solution with a concentration of 1 μM.

From sample 3D a fraction of 0.987 ml was added to 2.5 ml destilledwater, 1 ml of 5 times concentrated IMDM, and 0.5 ml of FCS, thusresulting in a solution with a concentration of 1 μM.

All fractions were filtered through a 0.22 μM filter before use.

The results are represented in tables and attached hereto. Sample 3A isrepresented in Tables 3 and 4 in FIG. 4, Sample 3B in Tables 5 and 6 inFIG. 5, Sample 3C in Tables 7 and 8 in FIG. 6, and Sample 3D in Tables 9and 10 in FIG. 7.

Results of biologic testing on Samples 3A through 3D.

Only Sample 3D showed a dose dependent inhibition of replication of bothvirus isolates under study. Inhibition was complete (100%) at aconcentration of 0.125 μM.

Sample 3D, originally consisting of 132 ml, of which almost 1 ml wasused in the biologic test system, was further analysed by HPLC in a TFAsystem.

The TFA-HPLC system comprises a nucleosil 10C18 column having a lengthof 150 mm and a diameter of 2 mm in an HP 1090M apparatus, whereby theflow is at a 350 μl/min rate. The eluens is a gradient of 100% A/0% B to100% B and 0A in 120 minutes whereby eluens A is 0.05% TFA(trifluoroacetic acid) in destilled water and eluens B is 0.03% TFA inacetonitrile.

This sample, consisting of ca. 130 ml, was lyophilized, after retaining1400 μl. The lyophilized substance was resuspended in 1 ml destilledwater and centrifuged. 0.1% of the supernatant was examined on theanalytical HPLC (result see graph 95E038 in FIG. 8), and 10% of thesupernatant was separated following preparative HPLC (result see graph95E043 in FIG. 9). Five fractions were isolated, lyophilized,resuspended in water, and lyophilized again. These fractions were named3D1, 3D2, 3D3, and 3D4 (see FIG. 10), fraction 3D5 was stored at −20° C.Samples of fractions 3D3 and 3D4 was analysed by sequence analysis, nosequence was detectable in either fraction.

Fractions 3D1, 3D2, 3D3 and 3D4 were presented for testing at thebiologic test system as described hereabove. Estimation of fractionquantities after HPLC, and based on the chromatogram, were:respectively: 120 μg, 30 μg, 30 μg, 50 μg and 50 μg.

Results of the test on HIV replication inhibition with these fractionsare represented in Tables 11 to 18 (FIGS. 1-18).

It was concluded, that subfractions 3D3 and 3D4 showed an inhibitoryeffect on the replication of the two primary HIV isolates. Inhibition bysubfraction 3D3 was complete at a concentration of 0.25 μM. Inhibitionby subfraction 3D4 was complete at a concentration of 0.063 μM.

Fraction 3D3 and 3D4 were further analyzed by HPLC.

Therefore, from Sample 3D, (as specified hereabove), two time 10% wastaken as starting material. Two preparative HPLC (same technique asbefore) runs were performed (see chromatogram 95E)58-2, FIG. 19). 10fractions were separately isolated (no's 1 through 10 ) within 22 and 32minutes elution time (see chromatogram with fractions in FIG. 20). Fourout of these fractions, no's 6, 7, 8 and 9 were lyophilized, resuspendedin destilled water and again lyophilized and were again presented fortesting in the biologic system as described hereabove.

Estimation of fraction quantities gave: 1 μg, 39 μg, 7.5 μG and 2.5 μgrespectively. Three fractions (6, 8 and 9) were solved in 1 ml medium(IMDM)+10% FCS, thus resulting in stock solution of 0.2 μM for fraction6, 1.5 μM for fraction 8, and 0.5 μM for fraction 9. 50 (1 of thesestock solution were added to 100 μl medium, providing finalconcentrations of 0.067 (M for fractions 6, of 0.5 μM for fraction 8,and of 0.17 μM for fraction 9. A dilution range was made only fromfraction 7 (0.5 μM, 0.25 μM, 0.125 μM and 0.063 μM).

These four fractions were now renamed: α, β, γ and δ respectively.

Results were: complete (100%) HIV replication inhibition for fractionsα, β, γ and δ at the concentration as tested. A dose dependentinhibition was observed for fraction β, being complete at aconcentration of 0.5 μM (see Tables 19 to 22 in FIGS. 21-24).

In a further experiment, it was demonstrated, that from fraction β, anumber of fractions could be isolated. Therefore, fraction 3D4β, asisolated from HPLC chromatography 95E058-2, was passed on a RP-HPLC inammonium acetate.

The HPLC step comprises the same column as described for the TFA-HPLC atthe same flow rate, the eluens is also a gradient running from 100% Aand 0% B to 100% B and 0% A in 120 minutes, whereby A and B in thissystem are 0.1% ammonium acetate in destilled water at pH=6 (A) and 100%acetonitrile (B).

The result of this new chromatography is displayed in chromatogram:95E070 (see FIG. 25).

Mass spectrometry of the fractions 3d3 and 3d4 (subfractions 1 through10) showed molecules with masses of between 400 and 500 Dalton.

Subfractions 4, 5, 6, 8 and 10 were presented to be tested in the samebiologic testing system as described above.

Test results showed low HIV replication inhibition activity forsubfractions 4, 5, 6 and 10 up to a concentration of 0.06 μM.Subfractions 8 showed a dose-dependent inhibition of HIV replicationwith 100% inhibition for HIVams55 and 90% inhibition for HIVams37 at aconcentration of 0.046 μM.

Subfraction 8 underwent a series of tests for mass spectrometry. It wasdetermined, that this subfraction consisted of a mixture of twomolecules with masses of 426 and 440 D.

ES/MEMS measurements for subfraction 8 ⁴²⁶ and 8 ⁴⁴⁰ showed positiveproductions of the following masses: both had a group of 120 D, a groupof 17 D, a group of 18 D, and a group of 11 D. They differed in a groupof 42 D for 8 ⁴²⁶, versus a group 56 D for 8 ⁴⁴⁰. No negative ions weredetected.

Mass spectrometry of other fractions in Samples 3d3 and 3d4(subfractions 1 through 10) showed molecules with masses ofapproximately 500 Dalton.

An Electrospray High Resolution Mass Spectrometry (ES-HRMS) was executedon the substances which showed HIV replication inhibition (subfraction8, Siliavir). These were defined earlier herein by methods forisolation, purification and biological testing.

10% of subfraction 8 from HPLC 95E085 (solubilized in 0.1% TFA) was usedtherefore.

Two separate molecules were found as main fractions. One of these twomolecules had the mass of 426.164300 D (subfraction 8 ⁴²⁶ orSiliavir⁴²⁶), the other of the two molecules with the mass of 440.1816 D(subfraction 8 ⁴⁴⁰ or Siliavir⁴⁴⁰). (See FIG. 26). The experiment wasexecuted with a Finnigan-MAT 900 instrument at the resolution power ofR=10,000. Consequently a sequential mass spectrometry (LC−MS)^(n) wasexecuted, and which confirmed earlier findings of ES-MSMS measurements.

Nuclear Magnetic Resonance (NMR)

NMR was executed on Siliavir with a Varian Unity 500 Mhz instrument. Asample was prepared from new base material for which raw leech headethanol extracts were prepared as before. The lyophilized base materialwas resuspended with 50 ml H₂O. A separative step was performed toseparate masses below and above 3kD. Therefore, this resuspendedsolution was repartitioned over 8 centriprep-3 (kD) tubes andcentrifuged 4 times. Filtrates from each filtration step were combined,lyophilized and consequently resuspended in 50% HAC.

Subsequently, as was described herein before, preparative HPLC wasperformed on the resuspended filtrate. Column 7.7×250 mm (Nuceosil10C18; flow adjusted at 2 ml/min; detection at 254 nm or 280 nm; TFAsystem; eluent A is 0.05% TFA; eluent B is 0.05% TFA in 100%acetonitrile; gradient: 0% B to 100% B in 60 minutes). The fraction atbetween 24.2 and 27 minutes was collected separately (see analyticalHPLC FIG. 27) and lyophilized.

This fraction, as separated, was resuspended in 500 μl 50% Hac, andpassed again at HPLC, with the ammonium acetate system. (Column 7.7×250mm: nucleosil 10C18, flow adjusted at 2 ml/min, detection at 254 nm;Ammonium Acetate (A.A.)-system; eluent A is 0.1% A.A. in H₂O at pH=6;eluent B is 100acetonitrile in 60 minutes).

As this sample showed a time dependent precipitation, it was centrifugedand supernatant and pellet were analysed separately (See FIG. 28 forchromatogram of supernatant, and FIG. 29 for chromatogram of the pellet,which was resuspended in 1.6 ml 50% HAC).

Fractions which eluted between 24 and 27 minutes from both thesupernatant passage on HPLC and the pellet passage on HPLC werecollected, combined and lyophilized. An analytical HPLC chromatogram ofthis collected material, consisting of both components Siliavir⁴²⁶ andSiliavir⁴⁴⁰, is shown in FIG. 30.

The material as described was prepared and resuspended with D₂O and 50%DAc for passage on the Varian Utility 500 Mhz instrument. Separately,the resuspended pellet from FIG. 29 hereabove was also separately passedon the same instrument. Different types of NMR-Spectra (Proton, C13,DQCosy, HSQc, and Noesy) were made. The results are shown in FIGS. 31-41for the spectra of both components together, while the results for theresuspended pellet (where one component, presumably Siliavir⁴²⁶, ispresent for 85% in the sample, and the other component, presumablySiliavir⁴⁴⁰, is present for only 15%) are shown in FIGS. 42-49.

Scanning Electron Microscopy/Energy Dispersing Spectroscopy (SEM/EDS)

The instrument used for this experiment was an (Akhashi) ISIDS-130.Preparation of the testmaterial was performed with both components(Siliavir⁴²⁶ and Siliavir⁴⁴⁰). The spectra showed peaks for C, N, and O.Moreover, a Si peak is shown, which probably derives from HPLC columnmaterial although it cannot be excluded that it derives from the activecompound. The presence of S in the spectrum can be attributed to aderivative of the compound, e.g. a salt, but may also be a pollution. NoP was found.

Results of NMR, MS and EMS/EDS and discussion

Discussed are the results in the NMR measurements on the test materialconsisting of the mixture of the two components (as shown in FIGS.31-41) and of the NMR measurements on the material consisting of 85% ofone component and 15% of the other component (as shown in FIGS. 42-49).

Singlet at 9.3 ppm correlates in HSQC at 148 ppm. This may be anisolated aromatic CH between 2 N's.

Two doublets at 6.75 and 7.54 ppm: 2±2 protons couple with 8.5 Hz. Thisconstant is comparable to tyrosine, and therefore points towards a parasubstituted aromatic ring.

This phenylfragment has two substituents, probably no direct C's asinteraction in Noesy spectrum is missing.

Two singlets at 3.5 and 3.7 ppm; 2±3 protonen. No DQCosy and/or Noesyinteractions. Correlations in HSQC at ca. 30 ppm. These are probabyN—Ch₃ groups, and probably not O—CH₃ groups. Because of their positionin the low field, a dimethylated hetero aromatic Z-ring system, e.g. axanthine-like fragment (a purine alkaloid), is a probable explanationfor these peaks in conjunction with the 9.3 ppm peak.

One triplet at 3.7 ppm: 2 protons. Correlates in DQCosy with a multipletat 1.72 ppm (2 protons); this multiplet correlates with the multiplet at1.46 ppm (2 protons).

One triplet at 3.4 ppm: 2 protons. Correlates in DQCosy with a multipletat 1.65 ppm (2 protons); this multiplet correlates with the samemultiplet at 1.46 ppm (2 protons).

It is concluded from these triplets and multiplets that this may pointtowards a Xl—(Ch₂)_(5—X)5 fragment, in which X₁ and X₂ are most probablyN, based on the chemical shift positions of the alpha Ch₂'s.

Signals at 1.2 and 1.4 ppm: probably artefacts or pollution.

Sulphur atoms are not likely to be present from these NMR spectra.

The NMR signals of the two components show that these are isomers. Theyonly differ 14.0173 D, which can only represent CH₂. Therefore, it isconcluded that Siliavir⁴⁴⁰ is equal to Siliavir⁴²⁶ plus CH₂.

The presence of the following groups is concluded or presumed:

A para-substituted aromatic ring, mass=76+Y₁+Y₂.

AB system in aromatic ring,

e.g. of the formula:

wherein Y₁ can for instance be —OH, —CH₂OH, —═C═O or —C(O)H, while Y₂can be an oxygen atom, a NH-group, an N-group substituted by C₁₋₃ alkylor C₁₋₃ (substituted) alkyl, C═).

A pentane fragment in the 426 component, possibly hexane in the 440component, e.g. of the formula:

wherein X₁ and X₂ may e.g. be: C, N, NCH₃, NSO₄, and X₃ to X₄ may e.g.be: H, NH₂, N═C═O or X₃+X₄, X₅+X₄, X₅+X₆, X₇+X₈, X₉+X₁₀, X₁₁X₁₂, X₁₃+X₁₄may together be an oxygen or a ═NH-group.

A methylated aromatic heterocyclic, e.g. xanthine-like fragment, withtwo CH₃ groups, e.g. of the type:

wherein Z₁, Z₂, Z₅, Z₇may e.g. be: C, H, CH₂OH, NCO₃; and Z₃, Z₄, Z₆, Z₈may e.g. be: C, H, CH₃, OH, CH₂OH.

A C—H between two N'S.

Based on the above presumptions the acive compounds may be compounds ofthe types:

wherein “O” is a carbon atom substituted with one or two hydrogen atoms.

Conclusion

It can be concluded, that HIV replication inhibition is strongly presentin a group of substances, which can be attributed in separate isolatesor molecules, or combination of molecules within this group. The groupof substances is described herein-above by methods for isolation andbiological testing.

The group of substances containing the HIV replication inhibitioncomprises e.g.:

3D3 and 3D4 as described. The molecules contained in this group ofsubstances for the major part have molecular weights of around 500Dalton.

Literature

Autrum H.: Bronn's Klassen und ordnungen des Tierreichs 1936 Berman P W.et al. : Nature 1990, 345: 622-625.

Blaise M.: J Med Vet Militaire 1874/5, 10.

Bucnocore L. & Rose J K: Nature 1990, 345: 625-628.

Harant H.: Arch Soc Sci Montpellier 1927, 10: 1-76 Jacks T. et al.:Nature 1988, 330: 280-283.

Jarry D.: Bull Soc Zoo1 Fr 1959, 84: 73-76.

Keegan H. L. et al. : Am J Trop Med Hyg 1970, 19-6: 1029-1030.

Kohl N E. et al: Proc Natl Acad Sci USA 1988, 85: 4686-4690.

Loeb D D. et al.: Nature 1989, 340: 397-400.

Mouquin-Tandon A. : Monographie de la famille des Hirunidees, Paris,1846.

Neveu-Lemaire.: Trate d'entomologie medicanale et veterinaire, 1938

Pauwels R. et al.: Nature 1990, 343: 470-474.

Rusche J R. et al.: Proc Natl Acad Sci USA 1985, 85: 3198-3202.

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Turner F M. : the Lancet 1969, 1400-1401.

Veronese F D. et al.: J Virol 1988, 62: 795-801.

What is claimed is:
 1. A method for obtaining an extract havinginhibiting activity toward a human immunodeficiency virus, comprising:preparing a suspension from tissue of a leech from the familyhirudinidae; fractionating the suspension; and selecting as the extracta protein-free fraction which exhibits a molecular weight in the rangeof 300 to about 600 Daltons and exhibits inhibiting activity towards ahuman immunodeficiency virus.
 2. The method of claim 1, whereinpreparing the suspension from tissue of a leech from the familyhirudinidae comprises extraction with a solvent comprising ethanol. 3.The method of claim 2, wherein the solvent comprises ethanol and water.4. The method of claim 1, further comprising: subjecting the fraction tofurther purification.
 5. The method of claim 4, wherein furtherpurification comprises: subfractionating said fraction to separatediscrete subfractions; and selecting as the extract a subfractionexhibiting inhibiting activity towards a human immunodeficiency virus.6. The method of claim 4, wherein further purification comprisesseparation on a HPLC column.
 7. The method of claim 4, wherein furtherpurification comprises separation on a HPLC column comprised ofnucleosil 10C18.
 8. The method of claim 1, further comprising:extracting leech tissue or secretions; lyophilizing the resultingsolution; suspending the lyophilized material in an aqueous solution;centrifuging the suspension to obtain a pellet and a supernatant;loading resulting supernatant on a size fractionation column; elutingthe column with an aqueous solution; saving fractions of the elutedmaterial; testing the fractions for anti-viral activity; and collectingas the extract the active fractions.
 9. The method of claim 8 whereinthe size-fractionation column comprises Sephadex g-75.
 10. The method ofclaim 1, further comprising: separating the fraction that exhibitsinhibiting activity towards a human immunodeficiency virus on a firstreverse phase HPLC column wherein the eluent is a gradient from asolution comprising trifluoracetic acid in water to a solutioncomprising trifluoracetic acid in acetonitrile; collecting a portion ofthe eluent corresponding to the major absorption peak of thechromatogram recorded at 254 nm; subjecting the portion tochromatography on a second reverse phase HPLC column wherein the eluentis a gradient from a solution comprising ammonium acetate in water to asolution comprising acetonitrile; and recovering an extract thatexhibits inhibitory activity towards human immunodeficiency virus bycollecting the eluent from the second reverse phase HPLC columncorresponding to the major absorption peak or peaks of the chromatogramrecorded at 254 nm.
 11. A method according to claim 1, wherein theextract has inhibitory activity in the micromolar range.
 12. A methodaccording to claim 11, wherein the extract completely inhibits the humanimmunodeficiency virus induced syncytium formation or p24 production at0.5 micromolar concentrations or less.
 13. A method according to claim12, wherein the extract has inhibitory activity of 0.17 micromolarconcentrations or less.
 14. A method according to claim 13, wherein theextract has inhibitory activity at 0.067 micromolar concentration. 15.An extract obtained by a method according to claim 1, wherein theextract exhibits a molecular weight between about 300 and about 600Daltons.
 16. The extract of claim 15, wherein the molecular weight isbetween about 400 and about 500 Daltons.
 17. The extract of claim 15,wherein the extract has inhibitory activity in the micromolar range. 18.The extract of claim 17, wherein the extract completely inhibits thehuman immunodeficiency virus induced syncytium formation of p24production at 0.5 micromolar concentrations or less.
 19. The extract ofclaim 18, wherein the extract has inhibitory activity at 0.17 micromolarconcentrations or less.
 20. The extract of claim 19, wherein the extracthas inhibitory activity at 0.067 micromolar concentration.
 21. Apharmaceutical composition comprising an extract obtained according toclaim 1, and a suitable vehicle for administration.
 22. A method ofinhibiting activity of a human immunodeficiency virus comprisingcontacting said virus with an extract obtained according to claim
 1. 23.A method for obtaining an extract having inhibiting activity towards ahuman immunodeficiency virus, comprising: preparing a suspension fromtissue of a leech from the family hirudinidae; fractionating thesuspension; and selecting as the extract a protein-free fraction whichexhibits a molecular weight in the range of 300 to about 600 Dalton andexhibits inhibiting activity towards a human immunodeficiency virus; andwherein said fraction is selected from the group of


24. A method of obtaining an extract having inhibiting activity toward ahuman immunodeficiency virus, comprising: preparing a suspension fromtissue of a leech from the family hirudinidae; fractionating thesuspension; and selecting as the extract a protein-free fraction whichexhibits a molecular weight in the range of 300 to about 600 Daltons andexhibits inhibiting activity towards a human immunodeficiency virus; andwherein said extract is of the formula Ar¹-spacer-Ar², wherein Ar¹ is asix ring aromatic which is substituted with one or more of a hydroxylgroup, an amino group, a C₁₋₄ alkyl group, or a halogenwherein—spacer—is a C₃₋₈ alkyl alkylene group coupled through ahydrophilic group to said Ar₁ group, and further wherein Ar² is aheteroaromatic 2-ringsystem of carbon and nitrogen atoms, said2-ringsystem having form 9 to 10 atoms in a purine-like ornaphthalene-like ringsystem, and said 2-ringsystem is substituted withone or more of a C₃₋₈ alkyl group, a hydroxy group, an amino group, or ahalogen group.